Project Summary Epstein-Barr virus (EBV) is a human gammaherpes virus that infects approximately 95% of the population and remains latent in memory B cells as a chromatin-associated multicopy episome. EBV infection is causally associated with several pathologies including different types of lymphomas and gastric carcinoma. This heterogeneity in EBV-associated diseases may reflect the different gene expression programs that the virus adopts in different cell types and host-cell conditions. We and others have shown that epigenetic modifications contribute to establish and maintain these alternative viral gene expression programs during latency. We identified that cellular factors such as CTCF affect the EBV epigenome; however, we still lack a complete understanding of mechanisms regulating EBV latency. Interactions with different nuclear sub-compartments, including the nuclear lamina, contribute to regulating the expression of underlying DNA regions. The nuclear lamina is a protein meshwork that underlies the inner nuclear membrane. In addition to providing structural support for the nucleus, the nuclear lamina is crucial for the proper organization of chromatin at the nuclear periphery. As such, genes that are localized to the nuclear periphery are frequently poorly expressed. Since CTCF is known to associate with the nuclear lamina, this supports the idea that the nuclear lamina contributes to the regulation of EBV gene expression. However, the role of the nuclear lamina as key regulator of the EBV epigenome has yet to be explored. We report here, for the first time, a potential interaction between the nuclear lamina and the EBV chromosome, which may regulate viral chromatin. At the present it remains unknown if EBV localizes at the nuclear periphery and how this association can affect viral genome functions. Our project aims to fill this gap by exploring the role of nuclear lamina-EBV interaction on the epigenetic regulation of the EBV genome. We hypothesize that the nuclear lamina contributes to the epigenetic control of EBV gene expression during latency, and that CTCF plays a role in the EBV-nuclear lamina interaction. Here we will use a genomic approach in EBV mutants and different EBV positive B-cell lines to: 1) identify which EBV genomic regions are associated with the nuclear lamina; the chromatin state of these lamin-associated regions; and the role of CTCF in mediating the interaction between lamins and the EBV genome; and 2) determine the role of nuclear lamina association on EBV gene expression and EBV chromatin organization. Our studies will provide new insight into the mechanism regulating EBV latent gene expression. Considering the role of nuclear lamina in the infection cycles of other viruses our proposal can have significant biological and translational implications for the broader virology field.